The present invention relates generally to videomicroscopy and more particularly to a novel method for adjusting a polarizing or interference type microscope and video camera in a video microscope system so as to improve the resolution, contrast, speed or recording, image fidelity and visibility of fine detail.
Video microscope systems are well known in the art and have been used in research and industry for over thirty years for examining various characteristics and properties of small objects. These systems generally include a microscope for forming an optical image of a specimen to be examined, a video camera for converting the optical image into a video image, a video monitor for displaying the video image and usually some form of recording device for permanently recording the video image in either analog or digital form. In U.S. Pat. No. 4,068,263, there is disclosed an example of a video microscope system which includes a microscope, a video camera and a video monitor. In U.S. Pat. No. 4,176,376, there is disclosed an example of a video microscope system in which electrical signals from a video camera are converted into digital signals and stored in a computer.
In the past, the video components in a video microscope system (i.e. the video camera and the video monitor) have been used merely as an alternative arrangement for either viewing an image appearing in the microscope or forming an image which can be recorded for scientific documentation rather than as a means for improving the quality of the image. In particular, the microscope has been adjusted to produce the best optical image of the specimen under examination and the video camera has been employed simply to produce a video image corresponding to the optical image so formed. Consequently, the image appearing in the video monitor has been at most comparable to the image appearing in the microscope and in many cases somewhat inferior.
One type of microscope which is very often used in combination with a video camera system is the polarizing microscope. Although the polarizing microscope has been in use for over a century to examine motile cells, its use in the study of non-muscle cell motility was quite limited until the 1950's when studies were undertaken to determine the factors that limit its sensitivity. These studies culminated in the developement of a polarizing microscope designed for maximum sensitivity. The heart of this instrument is a set of polarization rectifiers, one for each objective and condenser. Polarization rectifiers abolish the polarization cross visible in the rear focal plane of the objective by restoring the uniplanar state of polarization in the specimen and rear-focal planes. Rectification greatly enhances the sensitivity of polarizing microscopes by increasing the available contrast (measured as extinction factor) by as much as two orders of magnitude. These rectifiers also eliminate a troublesome diffraction anomaly causing spurious image contrast and resulting from the perturbing effect of the polarization cross in the Fourier transforms performed by the objective lens in image formation.
High-extinction polarized light microscopy has made possible many important advances in cell biology. However, polarization rectifiers are difficult and expensive to manufacture, and therefore only a limited number have been produced. Consequently, high-extinction polarized light microscopy has been applied by a very limited number of laboratories.
The present invention provides a powerful new method of polarized light microscopy which employes video-enhanced contrast. This method not only avoids the use of polarizing rectifiers entirely, but requires less stringent quality control over some optical components, as the deleterious effects of stray light arising from depolarization are minimal. Nevertheless, the new method matches or exceeds high-extenction methods in both sensitivity and resolving power and makes possible gains of up to four orders of magnitude in recording speed. The new method is based on video manipulations which allow amplification of image contrast while limiting the overall video image brightness.
Another type of microscope which has also been used extensively in combination with a video camera system is the differential interference contrast microscope.
Differential interference contrast (DIC) microscopy was introduced over a quarter of a century ago for the study of phase objects, and began to gain in popularity when it was shown to provide images of higher quality and with fewer artifacts than either phase-contrast or image-duplication interference microscopy. Images in the DIC microscope exhibit shadowcast details wherever interfaces (at organelles, membranes, fibrils, etc.) introduce gradients of optical path. The steepness of shadows depends on the difference in the refractive indices of a detail and its surround, the shape of the phase detail, and the bias retardation.
The strategy for optimizing visual and photographic contrast is well known: The compensator is manipulated, usually well within the range of .+-.0.1.lambda. from extinction to the desired setting, at which the dark line across the center of each specimen detail at extinction is shifted to one edge of that detail while the other edge brightens. To achieve the shadowcast effect of a differential image, the bias (or instrumental) retardation applied by the compensator adds to the retardation caused by the positive phase gradient on one side of the specimen and subtracts from that due to the negative phase gradient on the other side. Shadowing contrast is therefore directional, and different specimen details (phase gradients) are selectively emphasized at different settings of the compensator and at different specimen orientations. Under conditions that are optimal for viewing and for photomicrography, the images are severely light limited, especially at high magnification, and photomicrographic exposures of up to 20 seconds are required for exposure in fine grain negative, even with a bright, filtered mercury arc source. In addition, the full resolving power of the microscope usually cannot be employed, because resolution must be sacrificed to enhance contrast by partly closing the iris diaphragm. In accordance with this invention, the performance of the DIC microscope is dramatically improved. To realize this improvement, the iris diaphragm is opened to match the numerical aperture of the objective, and the bias retardation is increased to .lambda./9-.lambda./4, well beyond the usual .+-..lambda.20 range of a Brace-Kohler compensator. When the compensator is operated within the quasi-linear portion of the sine square curve, which forms the basis for contrast generation in all polarizing and interference microscopes, the visual and photographic images obtained are so saturated by stray light that the specimen may not be detectable to the eye or recordable on film. However, the video images and videomicrographs (photographs of the monitor) obtained by the method of this invention are superior in sharpness and detail to photomicrographs recorded under the very best of high extinction conditions.